GFP fusion proteins and their use

ABSTRACT

The present invention provides fusion proteins including a green fluorescent protein inserted into the internal amino acid sequence of a Gαs protein and further provides method of using the fusion protein construct to follow activation of a G-protein receptor by a candidate drug.

This application is a national stage application of PCT/US02/21484 filedJul. 3, 2002, which claims priority to U.S. Ser. No. 60/303,622 filedJul. 6, 2001.

This invention was made with government support under MH39595-10,AG15482 and awarded by the National Institute of Health (NIH). TheGovernment has certain rights in the invention.

The present invention relates a protein that is constructed by adding agreen fluorescent protein designated GFP that is internal to the aminoacid sequence of a G protein, in particular the Gαs protein. Theresulting fusion protein is a non-radioactive marker used, for example,for high throughput screening of G protein-coupled receptor drugtargets.

BACKGROUND OF THE INVENTION

A family of heterotrimeric nucleotide-binding proteins that bind toguanine (G proteins) transduces chemical and sensory signals across theplasma membrane by sequential interactions with receptor and secondmessenger-generating effectors. Because of the wide array of cellularprocesses that are mediated by G proteins, the study of G proteinfunction and regulation is a significant area of research in the signaltransduction field. There are reports containing suggestions of animportant function for G protein at cellular locations other than theplasma membrane. Certain G proteins were detected at intracellularmembranes, for example, the Golgi complex, whereas others associate withcytoskeletal structures, for example, microtubules and microfilaments.The mechanisms that govern the cellular destinations of G proteins andthe relative proportions of G proteins that traffic to subcellularcompartment are just beginning to be revealed.

G proteins are reported to couple the receptors for hormones orneurotransmitters to intracellular effectors such as adenylyl cyclase orphospholipase C. Twenty forms of the α-subunit of G proteins wereidentified and each is involved in the conveyance of multiple hormonalneurotransmitter signals from the outside of the cell to the effectsthat those hormones and neurotransmitters have on the inside of thecell.

G proteins may leave the membrane in response to neurotransmitter orhormone signals, but this has been very difficult to prove.

GFP, an autofluorescent protein of 238 amino acids, is a reportermolecule useful to monitor gene and protein expression and to observethe dynamics of protein movements within the living cell. Fusing GFP toanother protein of interest allows time-course studies to be performedon living samples in real time. Accounts of GFP fusion proteins includereceptors, secretory proteins, cytoskeleton proteins and signalingmolecules. Presently, GFP fusion proteins are constructed by generatingan expression construct that contains GFP fused in frame to either theN-amino or C-carboxyl terminus of the protein of interest. However, thisattachment may alter the function of the protein fused with GFPconsequently may not give results reflective of the natural state.

SUMMARY OF THE INVENTION

Fusion of a GFP protein at either NH₂ or COOH ends of Gαs proteinsubunits is not acceptable because the NH₂ region is important forassociation with Gαs protein βγ subunits, and the COOH terminal isrequired for interaction with receptors. Consequently, a biologicallyactive Gαs-GFP that incorporated GFP at some other positions of themolecule was developed. Suitable regions for insertion of a GFP sequenceare those regions that are free of interactions with receptors oreffectors.

A fusion protein was constructed by inserting an amino acid sequence ofa green fluorescent protein designated GFP, into the interior of anamino acid sequence of a G-protein, in particular the Gαs protein.Although, green fluorescent proteins have been inserted at either end ofG-proteins, a method was needed to insert GFP into the internal aminoacid sequence of a G-protein without altering the biological activity ofthe protein.

Green fluorescent protein (GFP) was inserted within the internal aminoacid sequence of Gαs to generate a Gαs-GFP fusion protein. The fusionprotein maintained a bright green fluorescence and was also identifiedby antibodies against Gαs or GFP, respectively. The cellulardistribution of Gαs-GFP was similar to that of endogenous Gαs. Gαs-GFPwas tightly coupled to the β adrenergic receptor to activate the Gαseffector, adenylyl cyclase. Activation of Gαs-GFP by cholera toxincaused a gradual displacement of Gαs-GFP from the plasma membranethroughout the cytoplasm in living cells. Unlike the slow release ofGαs-GFP induced by cholera toxin, the β adrenergic agonist isoproterenolcaused a rapid partial release of Gαs-GFP into the cytoplasm. At 1 minafter treatment with isoproterenol, the extent of this Gαs-GFP releasefrom plasma membrane was maximal. Translocation of Gαs-GFP induced byisoproterenol suggested that the internalization of Gαs might play arole in signal transduction by interacting with effector molecules andcytoskeletal elements at multiple cellular sites.

Uses for the Gαs-GFP fusion construct of the present invention include:

1. G proteins from the intracellular plasma membrane move in response toactivation by an antagonist. Following the activation of a G protein anddiscovering the time course for that activation. The occupancy of areceptor by an agonist is only the first step in a signaling cascade.The intracellular processes might be activated at different rates or, atspecific areas within a cell. Gαs-GFP is useful because it can befollowed in real time as events take place without disrupting naturalprogress of events.

2. Tracking protein functions in living cells.

3. As a non-radioactive marker for high throughput screening ofG-proteins coupled receptor drug targets, following the course ofactivation of a putative receptor or a putative ligand. For example, ifa drug company has a candidate that it believes activates G proteincoupled receptors in a functional sense, the Gαs-GFP fusion construct isuseful as a high throughput screen, because a change in fluorescence inresponse to the application of an agonist is detectable. Conversely, theactivity of an antagonist is visualized by adding it in 96 well plates,and screening significant numbers of samples on a fluorimeter todetermine which compounds block the expected fluorescence change.Gαs-GFP could be used in combination with a fluorescent receptor such asthat developed by the Biosignal Corporation in Montreal. To do this,cells are transfected with fluorescent receptors and Gαs-GFP. A ligandwhich activated the receptor in such a way that the G protein was alsoactivated should decrease the fluorescence of GFP induced by the emittedlight from the receptor (fluorescence resonance energy transfer-FRET).Thus, a number of candidate compounds may be screened for receptor and Gprotein activation by conducting these assays in e.g. 96 well plates.

4. The use of green fluorescent protein (GFP) in the study of cellularsignaling allows not only the observation of G protein trafficking, butthe opportunity to study the dynamics of G proteins in real time as wellas their function.

Other molecules may be modified in the same way, for example the otherof the 20 G protein α subunits. Insertion sites for GFP are determinedby an analysis of the sequence. None of the Gαs can be modified byadding GFP to either the amino or carboxy terminus because theirfunction would be destroyed. Putting the GFP in the internal regionsdoes not harm the effects of the protein, but rather bestows on its newproperties. Several other signaling molecules may be suitable candidatesfor the fusion proteins of the present invention.

5. Gαs-GFP is modified in such a way that it will couple to otherreceptors. Modification of amino acids near the carboxy terminalgenerates a fluorescent Γα that is capable of coupling to receptorswhich normally couple to Gαi, Gαo or Gαq (Conklin, et al., 1996). Thiswill allow the same fluorescent G protein to assess potency and efficacyof putative agonists and agonists for a large number of G proteincoupled receptors.

The 5 C terminal residues of Gαs are QYELL (SEQ ID NO: 3). They arereplaced with DCGLF (SEQ ID NO: 4) for Gi1 or Gi2, with ECGLY (SEQ IDNO: 5) for Gi3, with RCGLY (SEQ ID NO: 6) for Go, and with EYNLV (SEQ IDNO: 7) for Gq.

COS1 or HEK293 cells are suitable because they are easy to transfect.These or comparable cells are co-transfected with GFP-Gαs (either in itsnative form or engineered to couple to a receptor which normally couplesto Gi or Gq) and the desired receptor. Putative agonists are screened bymonitoring loss of fluorescence from the membrane. High-throughputfluorescence monitoring instruments that are known to those of skill inthe art are used for this purpose. Putative antagonists are screened byassessing their ability to block the effects of known receptor agoniststo evoke this phenomenon.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Gαs fusion protein cDNA construction. (A) shows a schematicof Gαs-GFP. Gαs-GFP defines the fusion protein in which GFP was insertedwithin the NH₂-terminal domain of the long Gαs. (B) presents a model ofGαs-GFP. The structure of GFP is shaded. The Gαs subunit structure isthat of Gαs-GTPγS.

FIG. 2 shows expression of the Gαs-GFP fusion in COS-1 cells. COS-1cells were lysed 24 h after transiently transfecting with Gαs-GFP (3 μgDNA). 30 μg protein was loaded, separated by SDS-PAGE gel, detected withpolyclonal antibody against Gαs (panel B) or monoclonal antibody againstGFP (Panel A), as indicated. Lane 1 represents a lysate from cellstransfected with Gαs-GFP, Lane 2 is the lysate from control cells.

FIG. 3 shows Gαs-GFP is associated with the plasma membrane intransfected cells. 24 h post-transfection, cells were observed byconfocal microscopy at 37° C. A computer-generated cross section of atypical cell is displayed on the top (x-z plane) and on the right (y-zplane). Each image shown is representative of at least 20 cellssubjected to a z-scan analysis. Similar results were obtained withCOS-1, PC12, and HEK 293 cells.

FIG. 4 shows subcellular distribution of Gαs-GFP in COS-1 cells.Particulate and soluble fractions were isolated from cells transfectedwith Gαs-GFP constructs 24 h post transfection as described herein. 20μg protein was loaded, separated by SDS-PAGE gel and detected with apolyclonal antibody against the C-terminal peptide of Gαs. Lanes 1 and 2represent the soluble portion from the control cells or cellstransfected with Gαs-GFP, respectively. Lanes 3 and 4 indicate theparticulate fraction from control cells or cells transfected withGαs-GFP, respectively.

FIG. 5 shows Gαs-GFP binding to AAGTP. COS-1 cells were co-transfectedwith cDNA encoding Gαs-GFP (1 μg) and β-adrenergic receptor (4 μg). (A)Shows cell membranes prepared 24 h post-transfection and incubated with³²P AAGTP in the presence and absence of isoproterenol (as indicated).Proteins were resolved by SDS-PAGE and autoradiography. Results shownare from one of four similar experiments. (B) Presents densitometricanalysis of Gαs-GFP binding to AAGTP. Densitometric analysis of fourindependent experiments were carried out and displayed in densitometricunits. [Shown is the mean±Standard error, n=4, ** indicates significantdifference from control treated without ISO (P<0.01].

FIG. 6 shows Gαs-GFP activates adenylyl cyclase. Cells were transfectedwith GFP (control) or Gαs-GFP, respectively and assayed for cAMPformation in the presence or absence of isoproterenol (ISO: 50 μM) asindicated. (A) control cells in the absence of ISO. (B) Gαs-GFPtransfected cells in the absence of ISO. C. control cells with ISO. D.Gαs-GFP transfected cells treated with ISO. The values shown aremean±standard error of nine samples from three experiments. Identicallevels of Gαs-GFP in each group were determined by western blotting. **indicates significant difference from control cells treated without ISO;(P<0.01).

FIG. 7 demonstrates cholera toxin treatment translocates Gαs-GFP inliving PC12 cells. (A) 24 h post-transfection with Gαs-GFP, media wasreplaced as described in Methods and living cells were viewed byconfocal microscopy at 37° C. Cells were initially imaged (0 min),cholera toxin (3 μg/ml) was added and cells were observed for 1 h.Bar=10 μm. (B) computer-generated cross section of the whole cell aftercompletion of the one hour, is displayed on the top (x-z plane) and onthe right (y-z plane). Results shown are from one of four comparableexperiments. Observation of other cell lines (COS-1 and HEK 293) showedsimilar results for response to cholera toxin.

FIG. 8 shows isoproterenol-stimulated rapid internalization of Gαs-GFPin living COS cells. Cells were transfected with Gαs-GFP and observed 24h later at 37° C. with confocal or digital fluorescent microscopy. (A)cells were treated with or without isoproterenol (20 μM), and imageswere captured every 5 seconds (A video scan; showed COS-1 cell treatmentwith ISO for 2 min. and; shown control COS-1 cell for 2 min). Arrowsindicate areas where membrane-bound Gαs-GFP released from plasmamembrane significantly. Clusters of Gαs-GFP form subjacent to the plasmamembrane (indicates by open arrowhead). (B) Observation of Gαs-GFPrelease from plasma membrane using confocal microscopy. Arrows displayregions where Gαs-GFP released from plasma membrane significantly. Thearrowheads indicate the sites where the Gαs-GFP was inserted after the 2minutes time point. Bar=10 mm. These results are typical of 40 of 58cells observed during the course of 15 experiments. Approximately 70% ofthe cells showed internalized Gαs-GFP in response to isoproterenol[ISO]. Thirty percent did not show a significant response to thisagonist.

FIG. 9A-9C are cDNA (nucleotide) (SEQ ID NO: 1) and its complement andamino acid sequences (SEQ ID NO: 2) of the Gαs-GFP. The letters in a boxindicate the start codon for Gαs-GFP. The circled letters form the stopcodon for Gαs-GFP. A, G, T and C are abbreviations of Adenine, Guanine,Thymine and Cytosine, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Construction of Gαs-GFP

Full length cDNAs encoding Gαs were excised from the PcDNA-1 vector bydigesting with Sam I and Xba I restriction enzymes. The full length EGFPcDNA was obtained by PCR from the PEGFP-N3 using appropriate primers(sense 5′ GGAATTCATGAGCAAGGGCGAGGAACTG-3′ (SEQ ID NO: 8); antisense5′-GCTCTAGACGACTTGTACAGCTCGT-3′) (SEQ ID NO: 9) and adding restrictionsites to its cDNA (EcoR I at the initiation codon and Xba I at end ofcDNA). To insert the EGFP within the sequence of Gαs, the first fragmentof Gαs (from 1 to 71 amino acids) was amplified by PCR with restrictionsites for Kap 1 at initiation codon and EcoR I at end of the fragment.The cDNA of the fragment was cloned into PcDNA3 vector by the Kap 1 andEcoR 1 restriction sites using primers (sense5′GGGTACCATGGGCTGCCTCGGCAACA-3′ (SEQ ID NO: 10); antisense5′-GGAATTCGTCCTCTTCGCCGCCCTTCT-3′) (SEQ ID NO: 11). Modified 7 EGFP cDNAwas spliced into the first fragment of Gαs by EcoR 1 and Xba 1restriction sites on PcDNA3 to get the fusion cDNA sequence of the firstfragment of Gαs and EGFP. The second fragment of Gαs (from 82 to 394amino acids) was also obtained using PCR with appropriate primers. Thesense primer contained a part of a sequence overlapping with the 3′ endof EGFP (5′-CAGAGCTGGACAAGTCCAACAGCGATGGTGAGAA-3′) (SEQ ID NO: 12). Theanti-sense primer contained an additional Xba 1 restriction site(5′-GCTCTAGACGACTTGTACAGCTCGT-3′) (SEQ ID NO: 9) The presenion cDNAfragment described above was amplified by PCR. The Gαs-GFP fusionfragment and the second fragment of Gαs were also linked using PCRstrategy. The full length Gαs-GFP was cloned into PcDNA3 at Kap 1 andXba 1 restriction sites. All DNA manipulations, including ligations,PCR, bacterial transformation were carried out using proceduresdisclosed herein. Plasmid purification was done using “plasmidpurification kit” following the manufacture instruction (QIAGEN).

Ligation Protocol

1. In a 1.5 ml microfuge tube, cut 10 mL expression vector with thedesired restricted enzyme in a total volume of 20 mL for 2 h at 31° C.

2. Loading the sample into 1% agarose gel, run the gel applying avoltage of 100 V. Run the gel long enough to resolve the fragments ofinterest.

3. Turn off the power supply and remove the gel from the apparatus.

4. Using “Gel Extraction Kit” (QIAGEN), purify fragments from gel.

5. In 0.5 ml microfuge tube, mix the fragments of vector (0.03 mg) andrelevant inserts, add 5 mL 4′ ligation buffer (GIBCOBRL), incubating ina total volume of 20 mL at 14° C. overnight with 0.1 units T₄ ligase.

6. Take out 10 mL to transformation.

Polymerase Chain Reaction

1. In 0.5 ml thin wall tube mix the following ingredients.

10′ buffer (GIBCOBRL) 5 μL MgCl₂ (GIBCOBRL) 5 μL primer 1 1 μL primer 21 μL template DNA 0.5 μL 4 dNTP mix (GIBCOBRL) 10 μL H₂O 26.5 μL Taqpolymerase (GIBCOBRL) 1 μL 50 mL2. Spin down one time for 15 seconds and put one-drop mineral oil intube.3. Turn on the automated thermal cycler.4. First denature simples 2 min at 94° C., then run program for 35cycles.

-   -   Denature 90 seconds    -   Anneal 50 seconds at 58° C.    -   Extend 1 min at 72° C.        When cycles finish, 7 min perform extra-extend at 72° C.        5. Run gel and purify the DNA with “PCP Purification Kit”        (QIAGEN).        Transformation Protocol        1. Add 5 ml of LB medium (10 g tryptone, 5 g yeast extract, 10 g        NaCl in 1 L H₂O) to sterile 10 ml tube.        2. Scrape HB 101 bacterial cells (one colony) from stock plate        with loop. Transfer cells to medium and shake bacterial cells        off loop. Put the tube in shaking incubator at 31° C. for 12 h.        3. Spin down bacterial cells at 2000×g for 3 min at room        temperature.        4. Gently resuspend pellet of bacterial cells in 1 ml 50 mM        CaCl², incubate for 40 min on ice.        5. Spin down again at 2000×g for 3 min at 4° C. Resuspend pellet        of bacterial cells in 100 ml 50 mM CaCl².        6. In 1.5 ml sterile microfuge tube, add 10 mL ligated plasmid        vector, then mix it with 100 mL competent bacterial cells.        7. Incubate the mixture on ice for 20 min and then transfer tube        to 42° C. for heat shock for 30 seconds.        8. Take the mixture, and add to plate (with antibiotic), agar        side top incubating at 37° C. overnight.

Three Gαs-GFP fusion constructs were made and expressed in COS-1 cells.In the Gαs-NGFP expression vector, in which the GFP was spliced to theN-amino terminus of Gαs sequence, the fusion protein could not associatewith the plasma membrane of cells (see FIG. 1, FIG. 2A). The attachmentof palmitate at Cys-3 of Gαs is required both for its membraneassociation and for its ability to mediate hormonal stimulation ofadenylyl cyclase. A sequence motif that serves as a predictor for asubset of palmitoylated proteins is Met-Gly-Cys at the amino terminus ofa protein. This motif found in the Gi and Gαs subfamily of G-proteinsubunits and other proteins such as receptor tyrosine kinases. The GFPconnected with the amino terminus of Gαs may affect the palmitoylationof Cys-3. A GFP tagged COOH terminal of Gαs, Gαs-CGFP was alsoconstructed. Although this attached to the membrane, it did not respondto hormone activation.

Gαs exists as a short and a long splice variant. Compared with shortGαs, long Gαs contains an additional 15 amino acids inserted at position72 of the polypeptide chain, and there is an exchange of glutamate forapartate at position 71. Although there has been some indication thatsubtle differences between short Gαs and long Gαs exist, the generalfunction of the two forms is similar. No substantial difference in thefunction of the two forms has been detected. Furthermore, the yeast Gαs,GPA1, has an “extra loop” in this region as well. Levis et al. (1992)modified the long Gαs form at a site (residues 77-81) within the 15amino acid insert to confer upon it recognition by an antibody directedagainst a well-defined peptide of the influenza hemaglutinin (HA).Addition of the HA epitope did not alter the ability of wild type Gαs tomediate hormonal stimulation of adenylyl cyclase or to attach to cellmembranes. Given the possibility that this region was “inert”, aGαs-GFP2 fusion protein was constructed by replacing the residues(72-81) within the long Gαs with a GFP sequence (see FIG. 1). A westernblot of membrane and cytosolic fractions (FIG. 2B), probed with ananti-Gαs polyclonal or anti-GFP monoclonal antibody, shows that Gαs-GFP2is expressed in COS-1 cells with a distribution comparable to that ofintrinsic Gαs. These results indicate that the GFP in the Gαs-GFP2should not alter the attachment of Gαs to membranes. In addition, thefluorescence of GFP in Gαs-GFP2 is visual and stable with UVirradiation.

Based on the α-carbon model of the α-subunit of the retinal G-proteintransducin, the sequence within which the 15 amino acid insert islocalized in the long Gαs serves as a linker between the ras-like domainand the α-helical domain. The guanine nucleotide-binding site isembedded between these two domains. Thus, the change in this linkersequence might be expected to diminish the ability of binding to guaninenucleotides of Gαs. To study this, COS-1 cells were co-transfected withGαs-GFP2 and β-adrenergic receptor cDNA. COS1 membranes were incubatedwith the photoaffinity GTP analog ³²P AAGTP as in the presence andabsence of a beta adrenergic agonist. Labeling of membranes from thetransfected COS-1 cells was accomplished by incubating with 0.1 mM [³²P]AAGTP for 5 min at 23° C., followed by treatment with isoproterenol(ISO) for 3 min. Gαs-GFP2 in COS-1 bound [³²P] AAGTP in response to ISO(FIG. 3). This result dramatically and unexpectedly demonstrated thatthe insertion of GFP into the linker sequence between two domains of Gαsdoes not disrupt agonist-induced guanine nucleotide exchange.

Cholera toxin activates Gαs by directly ADP ribosylating arginine 201 ofGαs and inhibiting the intrinsic GTPase. Thus, cholera toxin locks Gαsin the activated state. After, cholera toxin-activated was no longerobserved at the plasma membrane, but instead it was distributedthroughout the cytoplasm. Increased solubility of Gαs may correlate withactivation-induced depalmitoylation of Gαs, but it is not absolutelyclear that the removal of the lipid group is necessary for cytosolictranslocation. FIG. 4 shows that the Gαs-GFP on the cellular membrane isinternalized gradually subsequent to treatment of cells with choleratoxin. Cholera toxin activation of Gαs-GFP also provides furtherevidence that the fusion protein is capable of normal pysiologicalfunction.

The physiologic consequences of β-adrenergic receptor activation of Gαswere observed by examining the response of Gαs-GFP cos1 cells toisoproterenol. The rapid translocation of Gαs from membrane to cytoplasmwas clearly delineated.

To determine whether Gαs-GFP was fully physiologically active, testswere performed to see if the fusion protein was capable of activatingadenylyl cyclase. By measurement of cAMP accumulation in COS-1 cellstransfected in different conditions, the overexpression of Gαs-GFP wasfound not to alter the base level of cAMP in cells. Isoproterenoltreated cells showed the cAMP production in Gαs-GFP cells to besignificantly higher than cells transfected with GFP-vector alone (FIG.6).

Thus, assay of subcellular distribution and signaling function shows invitro and in vivo that the GFP insertion into the Gαs amino acidsequence does not substantially affect normal function of Gαs. The studyindicates a new approach to constructing GFP fusion protein and thestudy of G protein molecular signaling transduction in cells.

DOCUMENTS CITED

-   Conklin, B. R., Herzmark, P., Ishida, S., Voyno-Yasenetskaya, T. A.,    Sun, Y., Farfel, Z. and Bourne, H. R. (1996) Carboxyl-terminal    mutations of Gq alpha and Gs alpha that alter the fidelity of    receptor activation. Mol. Pharmacol. 50: 885-890.-   Hugges, T. E., Zhang, H., Logothetis, D. E., Berlot, C. H. (2001)    Visualization of a functional Gaq-green fluorescent protein fusion    in living cells. J. Biol. Chem. 276: 4227-4235.-   Kallal, L. and Benovic, J. L. (2000) Using green fluorescent    proteins to study G-protein receptor localization and trafficking.    TiPS 21: 175-180.-   Levis, M. J. and Bourne, H. R. (1992) Activation of a subunit of Gαs    in intact cells alters its abundance, rate of degradation, and    membrane avidity. J. Cell Bio. 5:1297-1300.-   Sunahara, R. K., Tesmer J. J. G., Gilman, A. G. and    Sprang S. R. (1997) Crystal structure of the adenylyl cyclase    activator Gαs. Science 278: 1943-1947.

1. A fusion protein comprising a green fluorescent protein insertedbetween the GTPase and the helical domain of Gα_(S), wherein the Gα_(S)activates adenylyl cyclase and the fusion protein translocates from theplasma membrane into the cytoplasm upon activation.
 2. The fusionprotein of claim 1, wherein the insertion is at regions that are free ofinteractions with receptors or effectors.
 3. The fusion protein of claim1 modified for specific receptors by replacing amino acid residues atthe C terminal end of Gα_(s).
 4. A method for making a fusion protein,said method comprising: (a) obtaining a molecule having the amino acidsequence of a green fluorescent protein; and (b) inserting the moleculebetween the GTPase and the helical domain of Gα_(S).
 5. The method ofclaim 4 wherein the fusion protein has the amino acid sequence of SEQ IDNO:
 2. 6. A method to follow an activation of a G-protein receptor by acandidate drug said method comprising: (a) obtaining a Gα_(s) greenfluorescent fusion protein, wherein the green fluorescent protein isinserted between the GTPase and the helical domain of Gα_(S); (b)monitoring fluorescence of the fusion protein in response to thecandidate drug; (c) inferring from a change in membrane fluorescencewhether the drug is an agonist or antagonist; and (d) following theactivation of the G-protein receptor by the candidate drug.
 7. Thefusion protein of claim 1 comprising the amino acid sequence of SEQ IDNO:
 2. 8. The fusion protein of claim 1, wherein the fusion proteininternalizes.